Nanoparticle-biomolecule conjugates are used for various purposes, including producing detectable signals in chemical and biological assays, such as immunohistochemistry (IHC) and in situ hybridization (ISH) assays. The efficiency of bioconjugates for such processes is determined, at least in part, by the number of biomolecules that are conjugated to each nanoparticle and available for whatever a particular purpose requires. For example, and with reference to using protein nanoparticle conjugates for in vitro diagnostics, one factor that determines bioconjugate efficiency is how many protein molecules are conjugated to a nanoparticle and available for binding target molecules.
As a first step in assessing bioconjugate efficiency, determination of the average number of biomolecules conjugated to nanoparticle can provide information that is needed to optimize a particular application of a bioconjugate. For example, antibody quantification, combined with tissue staining results, could provide information about the number of antibody molecules conjugated to a nanoparticle that provides the most specific and strongest staining in an IHC assay. An accurate method for determining biomolecule/nanoparticle ratios is highly desirable since it is not always the case that the more biomolecules conjugated to a nanoparticle the better.